Isolated nucleic acid molecule encoding peptides which form complexes with MHC molecule HLA-Cw*1601 and uses thereof

ABSTRACT

A family of tumor rejection antigen precursors, and the nucleic acid molecules which code for them, are disclosed. These tumor rejection antigen precursors are referred to as BAGE tumor rejection antigen precursors, and the nucleic acid molecules which code for them are referred to as BAGE coding molecules. Various diagnostic and therapeutic uses of the coding sequences and the tumor rejection antigen precursor molecules are described.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.08/196,630 filed Feb. 15, 1994, which issued on Nov. 4, 1997 as U.S.Pat. No. 5,683,886 which is a continuation-in-part of application Ser.No. 08/079,110, filed Jun. 17, 1993 which issued on Nov. 5, 1996 as U.S.Pat. No. 5,571,711.

FIELD OF THE INVENTION

This invention relates to nucleic acid molecules, proteins, and peptideswhich are useful in connection with the diagnosis and treatment ofpathological conditions. This invention further relates to said proteinsand peptides, which are processed to a peptide presented by the MHCmolecule HLA-Cw*1601, and the presented peptide itself. These peptidesare useful in diagnosis and therapeutic contexts.

BACKGROUND AND PRIOR ART

The process by which the mammalian immune system recognizes and reactsto foreign or alien materials is a complex one. An important facet ofthe system is the T cell response. This response requires that T cellsrecognize and interact with complexes of cell surface molecules,referred to as human leukocyte antigens ("HLA"), or majorhistocompatibility complexes ("MHCs"), and peptides. The peptides arederived from larger molecules which are processed by the cells whichalso present the HLA/MHC molecule. See Male et al., Advanced Immunology(J.P. Lipincott Company, 1987), especially chapters 6-10. Theinteraction of T cell and complexes of HLA/peptide is restricted,requiring a T cell specific for a particular combination of an HLAmolecule and a peptide. If a specific T cell is not present, there is noT cell response even if its partner complex is present. Similarly, thereis no response if the specific complex is absent, but the T cell ispresent. This mechanism is involved in the immune system's response toforeign materials, in autoimmune pathologies, and in responses tocellular abnormalities. Much work has focused on the mechanisms by whichproteins are processed into the HLA binding peptides. See Barinaga,Science, 257: 880 (1992); Fremont et al., Science, 257: 919 (1992);Matsumura et al., Science, 257: 927 (1992); and Latron et al., Science,257: 964 (1992).

The mechanism by which T cells recognize cellular abnormalities has alsobeen implicated in cancer. For example, in PCT applicationPCT/US92/04354, filed May 22, 1992, published on Nov. 26, 1992, andincorporated by reference, a family of genes is disclosed, which areprocessed into peptides which, in turn, are expressed on cell surfaces,which can lead to lysis of the tumor cells by specific CTLs. The genesare said to code for "tumor rejection antigen precursors" or "TRAP"molecules, and the peptides derived therefrom are referred to as "tumorrejection antigens" or "TRAs". See Traversari et al., Immunogenetics,35: 145 (1992); van der Bruggen et al., Science, 254: 1643 (1991), forfurther information on this family of genes. Also, see U.S. Pat. No.5,342,774.

In U.S. patent application Ser. No. 938,334, which issued on Apr. 11,1995 as U.S. Pat. No. 5,405,940 the disclosure of which is incorporatedby reference, nonapeptides are taught which are presented by the HLA-A1molecule. The reference teaches that given the known specificity ofparticular peptides for particular HLA molecules, a particular peptideis expected to bind one HLA molecule, but not others. This is important,because different individuals possess different HLA phenotypes. As aresult, while identification of a particular peptide as being a partnerfor a specific HLA molecule has diagnostic and therapeuticramifications, these are only relevant for individuals with thatparticular HLA phenotype. There is a need for further work in the area,because cellular abnormalities are not restricted to one particular HLAphenotype, and targeted therapy requires some knowledge of the phenotypeof the abnormal cells at issue.

In U.S. patent application Ser. No. 008,446, filed Jan. 22, 1993 whichhas been abandoned and incorporated herein by reference, it is disclosedthat the MAGE-1 expression product is processed to a second TRA. Thissecond TRA is presented by HLA-Cw*1601 molecule. The disclosure showsthat a given TRAP can yield a plurality of TRAs.

In U.S. patent application Ser. No. 994,928, filed Dec. 22, 1992, whichhas been abandoned and incorporated by reference herein, tyrosinase isdescribed as a tumor rejection antigen precursor. This referencediscloses that a molecule which is produced by some normal cells (e.g.,melanocytes), is processed in tumor cells to yield a tumor rejectionantigen that is presented by HLA-A2 molecules.

In U.S. patent application Ser. No. 08/032,978, filed Mar. 18, 1993,which issued on Apr. 15, 1997 as U.S. Pat. No. 5,620,886 andincorporated herein by reference, a second TRA, not derived fromtyrosinase, is taught to be presented by HLA-A2 molecules. The TRA isderived from a TRAP, but is coded for by a non MAGE gene. Thisdisclosure shows that a particular HLA molecule may present TRAs derivedfrom different sources.

In U.S. patent application Ser. No. 08/079,110 filed Jun. 17, 1993,which issued on Nov. 5, 1996 as U.S. Pat. No. 5,571,711 which isincorporated herein by reference, a new family of genes, referred totherein as the BAGE family, is disclosed. It was observed that thesegenes also code for tumor rejection antigen precursors. It is disclosedin the application that the MHC molecule known as HLA-Cw*1601 presents atumor rejection antigen derived from a BAGE tumor rejection antigenprecursor; however, the tumor rejection antigen was not disclosed. Thetumor rejection antigen is disclosed in U.S. patent application Ser. No.08/196,630 filed Feb. 15, 1994, which issued on Nov. 4, 1997 as U.S.Pat. No. 5,683,886 which is incorporated herein by reference. Theapplication also discloses ramifications stemming from the tumorrejection antigen, as well as therapeutic and diagnostic methodsutilizing the antigen.

The present application is directed to isolated nucleic acid moleculeswhich encode BAGE tumor rejection antigen precursors described in patentapplication Ser. No. 08/196,630 which issued on Nov. 4, 1997 as U.S.Pat. No. 5,683,886. The present application is further directed totherapeutic and diagnostic methods utilizing the isolated BAGE nucleicacid molecule.

The invention is elaborated upon further in the disclosure whichfollows.

BRIEF DESCRIPTION OF THE DRAWINGS

The above brief description, as well as further objects and features ofthe present invention, will be more fully understood by reference to thefollowing detailed description of the presently preferred, albeitillustrative, embodiments of the present invention when taken inconjunction with the accompanying drawings wherein:

FIG. 1 is comprised of FIGS. 1A through 1F. FIGS. 1A-C show lyticactivity of CTL clone 82/82 on MZ2-MEL sublines MZ2-MEL.3.0, MZ2-MEL.3.1and MZ2-MEL.B.TC.4. FIGS. 1D-F show lytic activity of CTL clone 82/82 onMZ2-MEL subline MZ2-MEL.43 and on melanoma cell lines MI4024/1-MEL andLB17-MEL, which were derived from HLA-Cw*1601 positive patients;

FIG. 2 shows TNF release by CTL 82/82 when put into contact with COS-7cells transfected with HLA-Cw*1601 alone, in combination with cDNA-AD5,or transfected with ADS alone. CTL 82/82 was also put into contact withMZ2-MEL.43 and MZ2-MEL.2.2.5, as controls;

FIG. 3 is comprised of FIGS. 3A through 3D. FIGS. 3A-C show lysis by CTLclone 82/82 of P1.HTR mouse cells cotransfected with expression vectorscarrying HLA-Cw*1601 and cDNA-AD5, as well as untransfected P1.HTR, andP1.HTR transfected with HLA-Cw*1601 alone. FIG. 3D shows lysis by CTLclone 82/82 of P1.HTR transfected with HLA-Cw*1601 and BAGE-derivednonapeptide AARAVFLAL; (SEQ ID NO: 3).

FIG. 4 sets forth nucleotide and amino acid sequences of a BAGE tumorrejection antigen precursor. The boxed segment is a tumor rejectionantigen derived from the precursor;

FIG. 5 represents Southern blots of DNA extracted from melanoma cellline MZ2-MEL.3.0, blood lymphocytes from patient MZ2 and mouse cell lineP1.HTR;

FIG. 6 represents Northern blot analysis of the expression of BAGE inMZ2-MEL.43 cells;

FIG. 7 represents PCR amplification of cDNAs from melanoma lines, tumorand normal samples, and of genomic DNA from subline MZ2-MEL.43; and

FIG. 8 represents lysis by CTL 82/82 of lymphoblastoid cell line MZ2-EBVincubated with BAGE-encoded peptide AARAVFLAL (SEQ ID NO: 3) or withnonapeptides ARAVFLALF (SEQ ID NO: 4) or MAARAVFLA (SEQ ID NO: 5).

DETAILED DESCRIPTION OF THE INVENTION EXAMPLE 1

Melanoma cell line MZ2-MEL was derived from patient MZ2 using standardmethodologies. This cell line is described in PCT ApplicationPCT/US92/04354, filed May 22, 1992, published Nov. 26, 1992, which isincorporated herein by reference. Once the cell line was established, asample of it was irradiated, so as to render it non-proliferative. Anumber of subclones were obtained from MZ2-MEL. Specifically, clonalline MZ2-MEL.3.0 was obtained from MZ2-MEL by limiting dilution. TheMZ2-MEL.3.0 culture was then cultured further. After more than 150generations in culture, a new subline, denoted MZ2-MEL.3.1, wasobtained. MZ2-MEL.3.1 was found to be resistant to a large fraction ofautologous CTL clones that had strong lytic activity on MZ2-MEL.3.0. Itwas determined that MZ2-MEL.3.1 had lost the genes coding for HLA-A29,B44, and Cw*1601 (see van der Bruggen et al., Eur. J. Immunol., 24:2134-2140 (1994), which is incorporated herein by reference).

Subline MZ2-MEL.43 was derived by limiting dilution from MZ2-MEL.3.0cells that has survived mutagen treatment (Van den Eynde et al., Int. J.Cancer, 44: 634-640 (1989)). Clonal subline MZ2-MEL.2.2, which does notexpress antigen MZ2-E, was selected from subclone MZ2-MEL.3.1 with anautologous anti-MZ2-E CTL clone (Van den Eynde et al., supra). SublineMZ2-MEL.2.2.5 was selected from subline MZ2-MEL.2.2 with an anti-MZ2-FCTL clone. MZ2-MEL.B.TC.4 was obtained by transfecting HLA-Cw*1601 geneinto subline MZ2-MEL.2.2.5 (van der Bruggen et al., supra). Melanomacell lines were grown as previously described by Van den Eynde et al.,supra and Traversari et al., Immunogenetics, 35: 145-152 (1992).

Cytolytic T cell clones ("CTLs") specific to cell line MZ2-MEL wereobtained utilizing irradiated MZ2-MEL cells. Specifically, a sample ofperipheral blood mononuclear cells ("PBMCs") was taken from patient MZ2,and contacted to the irradiated melanoma cells. The mixture was observedfor lysis of the melanoma cells, which indicated that CTLs specific fora complex of peptide and HLA molecule presented by the melanoma cellswere present in the sample.

The lysis assay employed was a chromium release assay following Herin etal., Int. J. Cancer, 39: 390-396 (1987), which is incorporated herein byreference. The assay, however, is described herein. The target melanomacells were grown in vitro, and then resuspended at 4×10⁷ cells/ml inDMEM, supplemented with DMEM with 10 mM HEPES and 50% FCS, and incubatedfor 60 minutes at 37° C. with 200 μCi/ml of Na(⁵¹ Cr)O₄. Labelled cellswere washed three times with DMEM, supplemented with 10 mM HEPES. Thesewere then resuspended in DMEM supplemented with 10 mM HEPES and 10% FCS,after which 100 μl aliquots containing 10³ cells, were distributed into96 well microplates. Samples of PBLs were added in 100 μl of the samemedium, and assays were carried out in duplicate. Plates werecentrifuged for 4 minutes at 100 g, and incubated for four hours at 37°C. in a 8% CO₂ atmosphere.

Plates were centrifuged again, and 100 μl aliquots of supernatant werecollected and counted. Percentage of ⁵¹ Cr release was calculated asfollows: ##EQU1## where ER is observed, experimental ⁵¹ Cr release, SRis spontaneous release measured by incubating 10³ labeled cells in 200μl of medium alone, and MR is maximum release, obtained by adding 100 μl0.3% Triton X-100 to target cells.

Those mononuclear blood samples which showed high CTL activity wereexpanded and cloned via limiting dilution, and were screened again,using the same methodology. The CTL clone MZ2-CTL 82/82 was thusisolated. The clone is referred to as "82/82" hereafter.

MZ2-MEL sublines and other melanoma lines were put into contact with CTLclone 82/82 and lytic activity was determined by measuring chromiumrelease. Chromium release was measured after 4 hours. FIG. 1A showslysis of MZ2-MEL.3.0 and MZ2-MEL.B.TC.4. Subline MZ2-MEL.3.1 was notlysed by CTL 82/82. FIG. 1B shows that there is lysis of clonal lineMZ2-MEL.43 by CTL 82/82. In addition, melanoma cell lines MI4024/1-MELand LB17-MEL, which carry the HLA-Cw*1601 allele, were lysed by CTL82/82.

EXAMPLE 2

The gene which codes for the antigen recognized by CTL 82/82 wasidentified. As described herein, the gene was identified bycotransfecting HLA-Cw*1601 cDNA with a cDNA library. Due to thespecificity of CTL 82/82 for MZ2-MEL.43, the cDNA of this cell line wasused to construct the cDNA library. In order to construct the cDNAlibrary from MZ2-MEL.43, poly-A+ RNA was extracted from MZ2-MEL.43 cellsusing an mRNA extraction kit. The mRNA was converted to cDNA usingrandom primers, ligated to adaptors following standard techniques, andinserted into the EcoRI site of expression vector pcD-SRα, whichcontains the replication origin of SV40. Recombinant plasmids wereelectroporated into E. coli JM101 and selected with ampicillin (50μg/ml). The library contained 66,000 inserts and was divided into 87pools of 400 bacteria and 297 pools of 200 bacteria. Each of these poolscomprised approximately 280 or 140 different cDNAs respectively, asabout 70% of the plasmids carried an insert. Each pool of bacteria wasamplified to saturation and plasmid DNA was extracted by the well knownalkaline lysis method.

Plasmid pcD-SRα was transfected with HLA-Cw*1601 cDNA. cDNA pools werethen cotransfected with the pcD-SRα plasmid containing HLA-Cw*1601 cDNAinto duplicate microcultures of COS-7 cells. Transfection was performedby the DEAE-dextran-chloroquine method (Seed et al., Proc. Natl. Acad.Sci. USA, 84: 3365-3369 (1987); Brichard et al., Annal. Biochem., 162:156-159 (1993); Coulie et al., J. Exp. Med., 180: 35-42 (1994)).Briefly, 1.5×10⁴ COS-7 cells were transfected with 100 ng of plasmidpcD-SRα containing CDNA of HLA-Cw*1601, and 100 ng of a pool of the cDNAlibrary or 100 ng of a cDNA clone. The HLA-Cw*1601 cDNA was isolatedfrom a cDNA library prepared with RNA extracted from subline MZ2-MEL.43(van der Bruggen et al., supra).

Cotransfectants were tested after 24 or 48 hours for their ability tostimulate the production of tumor necrosis factor (TNF) by CTLs(Traversari et al., Immunogenetics, 235: 145-152 (1992)). 1500 CTLs wereadded in 100 μl of Iscove's medium (Gibco BRL) containing 10% humanserum and 20 U/ml r-hu-IL-2 to microwells containing target cells. After24 hours, the supernatant was collected and its TNF content wasdetermined by testing its cytotoxic effect on cells of WEHI-164 clone 13(Espevik et al., J. Immunol. Methods, 95: 99-105 (1986)) in an MTTcolorimetric assay (Hansen et al., J. Cancer, 39: 390-396 (1989) andTraversari et al., supra).

Among the 384 pools of cDNAs (297 of 200 bacteria and 87 of 400) thatwere transfected, two produced positive supernatants containing morethan 40 pg/ml of TNF, whereas TNF concentration in all of themicrocultures transfected with the other pools was lower than 5 pg/ml.From one of these cDNA pools which contained 400 independent bacteria,800 bacteria were subcloned. Plasmid DNA was extracted from each of themand transfected into COS-7 cells together with the HLA-Cw*1601 constructdescribed supra. Twelve clones conferred recognition by CTL 82/82. Theresult obtained with one of them, denoted cDNA-AD5, is represented inFIG. 2.

FIG. 2 shows stimulation of CTL 82/82 by COS-7 cells cotransfected withCDNA-ADS and with an HLA-Cw*1601 CDNA, or transfected with one of thesecDNAs. The cDNAs were inserted in expression vector pcD-SRa as describedsupra. Samples of CTL 82/82 were added one day after the transfectionand the TNF content of the supernatant was estimated one day later bytesting its toxicity on cells of WEHI-164 clone 13. Positive andnegative controls were developed with MZ2-MEL.43 and MZ2-MEL.2.2.5cells.

To confirm the results obtained with cDNA-AD5 in transienttransfections, stable transfectants were also prepared. P1.HTR, a highlytransfectable variant derived from mouse tumor cell line P815 (Van Pelet al., Som. Cell Genet., 11: 467-475 (1985)), was transfected with bothHLA-Cw*1601 and cDNA-AD5, using the calcium phosphate precipitate methodwith plasmid pSVtkneoβ conferring resistance to geneticin (Nicolas etal., CSH Conferences Cell Prolif., 10: 469-485 (1983)) and HLA-Cw*1601alone, or both HLA-Cw*1601 and cDNA-AD5. The relevant cDNAs wereinserted in expression vector pcD-SRα as described supra. Clonalsublines were isolated from a geneticin-resistant transfectedpopulation. Transfected cells, when put into contact with CTL 82/82,were lysed by CTL 82/82, indicating that the antigen can also beprocessed in these mouse cells. FIG. 3A shows lysis by CTL clone 82/82of P1.HTR mouse cells cotransfected with expression vectors carryingHLA-Cw*1601 and cDNA-AD5. Untransfected P1.HTR and P1.HTR transfectedwith HLA-Cw*1601 alone were also tested.

EXAMPLE 3

DNA sequence analysis of cDNA-AD5 was performed by specific priming withsynthetic oligonucleotides. SEQ ID NO: 1 presents CDNA nucleotideinformation for the identified gene, referred to herein as "BAGE". Thesequencing reactions were performed by the dideoxy-chain terminationmethod (T7 Sequencing Kit, Pharmacia Uppsala Sweden,ΔTAq™Cycle-Sequencing Kit, USB, Cleveland, Ohio). The computer searchfor the sequence homology was done with programs FASTA@EMBL-Heidelbergand blast@ncbi.nlm.nih.gov.. The sequence bears no significantsimilarity to any other sequence presently recorded in databanks, exceptfor an Alu repeat (nucleotides 385 to 484) located outside of the codingregion.

EXAMPLE 4

The region of BAGE which codes for the antigen presented by HLA-Cw*1601was determined. In order to identify this region, a large number oftruncated BAGE cDNA clones were produced. By digesting BAGE withexonuclease III for various incubation times, progressive deletionsstarting from the 3' end were generated. The truncated variants werereligated into pcDNAI/Amp, electroporated into E. coli strain DH5αF'IQ,and selected via ampicillin (50 μg/ml). 438 clones were obtained in thisway.

The plasmid DNA was obtained from these 438 cones, and transfected intoCOS-7 cells together with HLA-Cw*1601 cDNA to test for their ability tocode for the antigen. The transfectants were tested in a TNF releaseassay, as described supra. Positive clones were those which stimulatedTNF release by CTL 82/82.

Once cells were divided into positive and negative transfectants, thesequences of plasmid DNA from 5 positives and 5 negatives weredetermined. Clone 19C2, a positive clone, contained part of the openreading frame for the BAGE gene described supra, from nucleotide 201 tonucleotide 267. In contrast, clone 17G12, a negative transfectant,contained nucleotides 201-206. This indicated that the antigenic peptidewas encoded by the first 67 nucleotides of the open reading frame.

FIG. 4, which shows the sequence of BAGE, also shows a putative proteinof 43 amino acids encoded by the largest open reading frame. Thisprotein was identified as containing the sequence of the presentedpeptide. The 43 amino acid protein, referred to herein as SEQ ID NO: 2,is as follows:

Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln Ala ArgLeu Met Lys Glu Glu Ser Pro Val Val Ser Trp Arg Leu Glu Pro Glu Asp GlyThr Ala Leu Cys Phe Ile Phe

The sequence corresponding to the peptide recognized in association withHLA-Cw*1601 by MZ2-CTL 82/82 is indicated in a box. The sequence isreferred to herein as SEQ ID NO: 3: Ala Ala Arg Ala Val Phe Leu Ala Leu.The sequence of primers VDB85 (SEQ ID NO: 6) (sense) and VDB86 (SEQ IDNO: 7) (anti-sense) used for PCR amplification, as discussed in Example4, are underlined with arrows.

Several synthetic peptides were prepared on this basis. Peptides weresynthesized on solid phase using F-moc for transient NH2-terminalprotection as described by Atherton et al., J. Chem. Soc. Lond. PerkinTrans., 1: 538-546 (1981) and characterized by mass spectrometry. Allpeptides were >90% pure as indicated by analytical HPLC. Lyophilizedpeptides were dissolved at 20 mg/ml in DMSO, diluted at 2 mg/ml in 10 mMacetic acid and stored at 80° C. Peptides were tested in a CTLstimulation assay with COS-7 cells transfected by HLA-Cw*1601 andincubated with the peptides. They were also tested by chromium releaseassay as previously described (Boon et al., J. Exp. Med., 152: 1184-1193(1980)). In this peptide sensitization assay, target cells were ⁵¹Cr-labelled for one hour at 37° C. and washed extensively. 1000 targetcells were then incubated in 96-well microplates in the presence ofvarious concentrations of peptide for 30 minutes at 37° C. before CTL82/82 cells were added. Chromium release was measured after 4 hours at37° C.

FIG. 8 shows lysis by CTL 82/82 of lymphoblastoid cell line MZ2-EBVincubated with BAGE encoded peptide AARAVFLAL (SEQ ID NO: 3). The finalconcentration of peptides during the incubation of the target cells withthe CTL is indicated. The arrow indicates the percentage of lysis ofMZ2-MEL.43 cells. Sensitization of a lymphoblastoid cell line frompatient MZ2 to lysis by CTL 82/82 was observed with nonapeptideAARAVFLAL (SEQ ID NO: 3) (amino acids 2-10, FIG. 4). Half-maximal lysiswas obtained at a peptide concentration of 30 nM (FIG. 8). Nonapeptidesthat did not include the N-terminal Ala, or ARAVFLALF (SEQ ID NO: 4) orthe C-terminal Leu, MAARAVFLA (SEQ ID NO: 5) were not able to sensitizetarget cells to lysis. P1.HTR cells were transfected with HLA-Cw*1601and were incubated with nonapeptide AARAVFLAL (SEQ ID NO: 3). Thetransfected cells were lysed by CTL 82/82. FIG. 3B shows lysis by CTLclone 82/82 of P1.HTR transfected with HLA-Cw*1601 and incubated with 1μM of the BAGE-encoded nonapeptide AARAVFLAL (SEQ ID NO: 3). Lysis ofchromium-labelled cells was tested after 4 hours.

From two MLTCs set up with different blood samples of patient MZ2, sixCTL clones that recognized the BAGE/HLA-Cw*1601 antigen were derived.They produced TNF in the presence of COS-7 cells cotransfected withHLA-Cw*1601 and BAGE cDNA-AD5. They also responded to cells transfectedwith HLA-Cw*1601 and incubated with nonapeptide AARAVFLAL (SEQ ID NO:3). It appears that at least 3 different CTL precursors can recognizethis BAGE antigen. CTL clone 82/1 expressed Vα2, Vα3 and Vβ13 while CTLclone 25/244 expressed Vα8 and Vβ8, whereas CTL clone 82/82 expressedVα3, Vα4 and Vβ13. Vα and Vβ expression were determined as follows:Total RNA from the different CTL clones was prepared by using RNAzol*B(Cinna/Biotecx, Friendswood, Tex.). Single-stranded CDNA synthesis wascarried with oligo(dT) and Moloney murine leukemia virus-derived reversetranscriptase without RNAse H activity. PCR was carried out byamplification of TCR-α and -β cDNA with the oligonucleotide primerscomplementary to TCR variable (Vα1-W29, Vβ1-W24) and constant (Cα, Cβ)region sequences described by Genevee et al., Eur. J. Immunol., 22:1261-1269 (1992). Specificity of TCR Vα and Vβ PCR amplification wasassessed by Southern blotting and hybridization with ³² P-labelled Cα orCβ oligonucleotides internal to the ones used for amplification.

EXAMPLE 5

The expression of BAGE in tissues was tested by reverse transcriptionand nested PCR (van der Bruggen et al., supra). cDNAs from melanomalines, tumor and normal tissue samples, and of genomic DNA from sublineMZ2-MEL.43, were amplified by PCR. Total RNA was extracted by theguanidine-isothiocyanate procedure as described by Davis et al., BasicMethods in Molecular Biology, pp. 130-135 (New York, Elsevier, 1986).Reverse transcription was performed on 2 μg of total RNA in a reactionvolume of 20 μl with 4 μl of 5×reverse transcriptase buffer, 2 μl of a20 mM solution of oligo(dT) 15 primer, 20 U of RNasin, 2 μl of 0.1Mdithiothreitol and 200 U of MoMLV reverse transcriptase plus 1 μl ofeach of 10 mM solution of dNTP. The reactants were incubated at 42° C.for 60 minutes. One twentieth of the cDNA product was then supplementedwith 5 μl of 10×thermostable DNA polymerase buffer, 1 μl each of 10 mMsolution of dNTP, 1 μl each of 25 μM solution of primers, 1 U ofDynaZyme™ and water to a final volume of 50 μl. The PCR primers were5'-TGGCTCGTCTCACTCTGG-3' (SEQ ID NO: 6) (VDB85, sense, nucleotide 100 to117) and 5'-CCTCCTATTGCTCCTGTTG-3' (SEQ ID NO: 7) (VDB86, anti-sense,nucleotide 367 to 385). PCR was performed for 30 cycles (1 minute at 94°C., 2 minutes at 62° C. and 2 minutes at 73° C.). 10 μl of the PCRproduct was size-fractionated on a 1.5% agarose gel. The quality of RNApreparations was tested by PCR amplification of human β-actin cDNA withprimers 5'-GGCATCGTGATGGACTCCG-3' (SEQ ID NO: 8) (exon 4, sense) and5'-GCTGGAAGGTGGACAGCGA-3' (SEQ ID NO: 9) (exon 6, anti-sense) for 21cycles of 1 minute at 94° C., 2 minutes at 68° C. and 2 minutes at 72°C. by AmpliTaq DNA polymerase.

PCR products were visualized on a 1.5% agarose gel stained with ethidiumbromide. No expression of gene BAGE was found in normal adult tissuesexcept in testis (see FIG. 7 and Table 1, below). The gene was alsosilent in placenta and umbilical cord and in several tissue samples fromfetuses older than 20 weeks. No expression of BAGE was found in thetwelve EBV-transformed lymphoblastoid cell lines tested nor in bloodlymphocytes stimulated with phytohemagglutinin.

                  TABLE 1    ______________________________________    Expression of Gene BAGE by    Normal Adult and Fetal Tissue    Adult tissues  expression    ______________________________________    Adrenal gland  -    Bone marrow    -    Brain          -    Breast         -    Cerebellum     -    Colon          -    Heart          -    Kidney         -    Liver          -    Lung           -    Melanocytes    -    Muscle         -    Ovary          -    Prostate       -    Skin           -    Sperm          -    Splenocytes    -    Stomach        -    Testis         +    Thymocytes     -    Urinary bladder                   -    Uterus         -    Placenta       -    Umbilical cord -    Benign naevus  -    Fetal tissues    Fibroblasts    -    Brain          -    Liver          -    Spleen         -    Thymus         -    Testis         -    ______________________________________

BAGE appears to be silent in normal adult tissues including melanocytes,except for testis. Because its expression was tested byreverse-transcription and PCR, the absence of a detectable product innormal tissues indicates a level of expression lower than 0.1% of thatobserved in tumor MZ2-MEL.

EXAMPLE 6

Expression of BAGE gene in tumor samples and cell lines was alsodetermined. Six hundred samples of tumors of various histologicalorigins were analyzed for BAGE expression. As shown in Table 2, below,BAGE gene is expressed mainly in melanomas (22%), bladder carcinomas(15%), mammary carcinomas (10%) and head and neck squamous cellcarcinomas (8%). A smaller proportion of positive samples was found insarcomas (6%) and in non-small cell lung carcinomas (6%). No expressionof BAGE was found in renal, colorectal and prostatic carcinomas,leukemias, or lymphomas. With very few exceptions, tumor samples thatexpressed BAGE also expressed one of the MAGE genes discussed generally,supra.

                  TABLE 2    ______________________________________    Expression of gene BAGE by tumor samples                      Number of BAGE    Histological type positive tumors*    ______________________________________    Melanomas         40/178    primary lesions   3/38    metastatic lesions                      37/140    Bladder carcinomas                      9/62    superficial tumors                      0/32    infiltrating tumors                      9/30    Mammary carcinomas                      8/79    Head and Neck squamous                      4/53    cell carcinomas    Lung carcinomas NSCLC°                      4/64    Sarcomas          1/18    Renal sarcomas    0/50    Colorectal carcinomas                      0/42    Prostatic carcinomas                      0/22    Leukemias and lymphomas                      0/22    ______________________________________     *Expression of gene BAGE was tested by RTPCR amplification of total RNA     with the primers shown on FIG. 4.     °NSCLC = non small cell lung carcinomas

BAGE was more frequently expressed in metastatic lesions of melanomas(26%) than in primary lesions (8%). In transitional-cell carcinomas ofthe urinary bladder, 30% of invasive tumors expressed BAGE, while noexpression was observed in superficial tumors. BAGE was expressed in ahigher proportion of tumor cell lines than of tumor samples: 32/60melanoma (53%) and 3/15 colorectal carcinoma cell lines (20%) werepositive. This has also been observed with MAGE genes, and may be due tothe fact that tumor cell lines are more readily derived from metastatictumors.

EXAMPLE 7

HLA-Cw*1601, the presenting molecule of BAGE antigen, cannot beidentified in serological assays, as useful antibodies are notavailable. However, its expression can be tested by reversetranscription and nested PCR. Approximately 7% (7/99) of Caucasianindividuals were found to express this HLA allele (van der Bruggen etal., supra). The concentration of HLA-C molecules on the cell surfacehas been reported to be about tenfold lower than that of HLA-A and B,possibly because of less efficient binding to β2-microglobulin (Neefjeset al., Eur. J. Immunol., 18: 801-810 (1988)). Nevertheless, it has bendetermined that BAGE codes for a peptide recognized on a HLA-C molecule,suggesting that HLA-C molecules also play a significant role in thepresentation of antigens to CTL.

EXAMPLE 8

A Southern blot with DNA extracted from blood lymphocytes of patient MZ2and from the melanoma cell line MZ2-MEL.3.0 was prepared. In order toperform Southern blot analysis, DNA from melanoma cell line MZ2-MEL.3.0,PBLs of patient MZ2 and mouse cell line P1.HTR were digested with EcoRIor HindIII. DNA capillary transfer was done by alkaline blotting on aZeta-Probe® membrane (Bio-Rad). Following transfer, the membrane wasrinsed in 2×SSC, baked for 1 hour at 80° C. and pretreated for 30minutes at 60° C. in 6×SSC, 10×Denhardt's solution. The membrane wasthen hybridized for 18 hours at 65° C. in 3.5×SSC, 1×Denhardt'ssolution, 25 mM NaH₂ PO₄ pH 7.0, 0.5% SDS, 2 mM EDTA, 100 μg/ml ofherring sperm DNA, and 2×10⁶ cpm/ml of a 121 bp ³² P-labelled probe(nucleotides 211 to 331 of SEQ ID NO: 1) produced by PCR. The membranewas then washed at 65° C. for 2×15 minutes in 2×SSC, 0.5% SDS, then for15 minutes in 0.2×SSC, 0.1% SDS, and autoradiographed for 10 days.

When this blot was hybridized with the 121 bp probe described supra,four bands were observed in lanes containing DNA digested with EcoRI and6 bands after HindIII digestion (FIG. 5). Considering the small size ofthe probe and considering the absence of EcoRI and HindIII restrictionsites in the coding sequence, these results indicate that BAGE belongsto a family of several related genes.

EXAMPLE 9

A Northern blot prepared with poly-A+ RNA of subline MZ2-MEL.43 washybridized with a 286 bp BAGE probe including nucleotides 100 to 385 ofSEQ ID NO: 1. To perform Northern blot analysis, poly-A+ RNA fromMZ2-MEL.43 was prepared using mRNA extraction kit. Total RNA from mousekidney tissue was extracted by the guanidine-isothiocyanate procedure asdescribed by Davis et al., supra. Poly-A+ RNA was purified from totalRNA on an oligo-dT column. For the Northern blot analysis, 5 μg ofpoly-A+ RNA from subline MZ2-MEL.43 and 5 μg of poly-A+ RNA from mousekidney cells were fractionated on a 1% agarose gel containing 0.66Mformaldehyde and transferred on a membrane in 10×SSC.

The membrane was pre-hybridized for 15 minutes at 60° C. in 10% dextransulfate, 1% SDS and 1M NaCl and hybridized overnight at 60° C. in thesame solution with 2×10⁶ cpm/ml of the 286 bp ³² P-labelled probe. Themembrane was washed at room temperature in 0.2×SSC for 10 minutes andthen 2×20 minutes at 60° C. in 0.2×SSC supplemented with 0.1% SDS, andautoradiographed for 15 hours. Control hybridization was performed onthe same membrane with a mouse β-actin probe.

FIG. 6 shows the results of this work. Each lane contained 5 μg ofpoly-A+ RNA from MZ2-MEL.43 cells. Control hybridization was performedon the same membrane with a β-actin probe. Two bands of approximately 1and 2.4 kb were observed.

Thus far, two main classes of antigens recognized by autologous CTL havebeen found on human melanoma. The antigens of the first class areencoded by genes that are expressed very specifically in tumors. Anantigen encoded by gene MAGE-1 was the first example (van der Bruggen etal., Science, 254: 1643-1647 (1991)), followed by other antigens encodedby genes MAGE-1 and MAGE-3 (Gaugler et al., J. Exp. Med., 179: 921-930(1994); van der Bruggen et al., supra). A tumor rejection antigenobserved on mouse mastocytoma P815 also resulted from the activation ofa gene which is silent in all normal adult tissues with the exception oftestis (Van den Eynde et al., J. Exp. Med., 173: 1373-1384 (1991)). Thesecond class of antigens represents differentiation antigens encoded bygenes that are expressed only in melanocytes and melanomas. Antigensencoded by tyrosinase were the first examples of this class (Brichard etal., Annal. Biochem., 162: 156-159 (1993); Robbins et al., Cancer Res.,54: 3124-3126 (1994); Wolfel et al., Eur. J. Immunol., 24: 759-764(1994)), which also comprises antigens encoded by Melan-A/MART-1 (Coulieet al., J. Exp. Med., 180: 35-42 (1994); Kawakami et al., Proc. Natl.Acad. Sci. USA, 91: 3515-3519 (1994)) and gp 100/pmel17 (Bakker et al.,J. Exp. Med., 179: 1005-1009 (1994); Cox et al., Science, 264: 716-719(1994)).

The foregoing examples show the isolation of a nucleic acid moleculewhich codes for a tumor rejection antigen precursor. This "TRAP" codingmolecule, however, is not homologous with any of the previouslydisclosed MAGE coding sequences described in the references set forthsupra. Hence, one aspect of the invention is an isolated nucleic acidmolecule which comprises the nucleotide sequence set forth in SEQ IDNO: 1. This sequence is not a MAGE coding sequence, as will be seen bycomparing it to the sequence of any of the MAGE genes described in thereferences. Also a part of the invention are those nucleic acidsequences which also code for a non-MAGE tumor rejection antigenprecursor but which hybridize to a nucleic acid molecule containing thedescribed nucleotide sequence, under stringent conditions. The term"stringent conditions" as used herein refers to parameters with whichthe art is familiar. More specifically, stringent conditions, as usedherein, refers to hybridization in 3.5×SSC, 1×Denhardt's solution, 25 mMsodium phosphate buffer (pH 7.0), 0.5% SDS, and 2 mM EDTA for 18 hoursat 65° C. This is followed by four washes of the filter at 2×15 minutesin 2×SSC, 0.5% SDS and 1×15 minutes in 0.2×SSC, 0.1% SDS at 65° C. Thereare other conditions, reagents, and so forth which can be used, whichresult in the same degree of stringency. The skilled artisan will befamiliar with such conditions, and thus they are not provided herein.

It will also be seen from the examples that the invention includes theuse of the sequences in expression vectors, as well as in thetransformation or transfection of host cells and cell lines, includingprokaryotic cell strains (e.g., E. coli), and eukaryotic cells (e.g.,CHO or COS cells). The expression vectors require that the sequence beoperably linked to a promoter. The expression vector may also include anucleic acid sequence coding for HLA-Cw*1601. Where the vector containsboth coding sequences, it can be used to transfect a cell which does notnormally express either one. The tumor rejection antigen precursorcoding sequence may be used alone, when, for example, the host cellalready expresses HLA-Cw*1601. Of course, there is no limit on theparticular host cell which can be used. As the vectors which contain thetwo coding sequence may be used in HLA-Cw*1601 presenting cells ifdesired, and the gene for tumor rejection antigen precursor can be usedin host cells which do not express HLA-Cw*1601.

The invention also includes expression kits, which allow the artisan toprepare a desired expression vector or vectors. Such expression kitsinclude at least separate portions of each of the previously discussedcoding sequences. Other components may be added, as desired, as long asthe previously mentioned sequences, which are required, are included.

To distinguish the nucleic acid molecules and the TEAPs of the inventionfrom the previously described MAGE family, the invention shall bereferred to as the BAGE family of genes and TRAPs. "BAGE" refers to thetumor rejection antigen precursors coded for by the previously describedsequence. "BAGE coding molecule" and similar terms, are used to describethe nucleic acid molecules themselves.

Also a part of the invention are peptides, for example, the peptide ofSEQ ID NO: 3, which can be used to identify those cells which presentMHC molecule HLA-Cw*1601. Administration of the peptides, carrying adetectable signal, e.g., followed by the identification of cells towhich the peptide has bound, is one way to accomplish this. Another wayto accomplish this is the use of solid phase bound peptides, to whichHLA-Cw*1601 presenting cells bind, thus removing them from the samplebeing assayed.

Additionally, the invention permits the artisan to diagnose a disordercharacterized by expression of the TRAP. These methods involvedetermining expression of the TRAP gene, and/or TRAs derived therefrom,such as the TRA presented by HLA-Cw*1601. In the former situation, suchdeterminations can be carried out via any standard nucleic aciddetermination assay, including the polymerase chain reaction, orassaying with labelled hybridization probes. In the latter situation,assaying with binding partners for complexes of TRA and HLA, such asantibodies, is especially preferred. An alternate method fordetermination is a TNF release assay, of the type described supra.

The isolation of the TRAP gene also makes it possible to isolate theTRAP molecule itself, especially TRAP molecules containing the aminoacid sequence coded for by SEQ ID NO: 1. These isolated molecules whenpresented as the TRA, or as complexes of TRA and HLA, such asHLA-Cw*1601, may be combined with materials such as adjuvants to producevaccines useful in treating disorders characterized by expression of theTRAP molecule. In addition, vaccines can be prepared from cells whichpresent the TRA/HLA complexes on their surface, such asnon-proliferative cancer cells and non-proliferative transfectants.Immunization against both BAGE and MAGE antigens can be undertaken. Inall cases where cells are used as a vaccine, these can be cellstransfected with coding sequences for one or both of the componentsnecessary to prove a CTL response, or can be cells which express bothmolecules without transfection. Further, the TRAP molecule, itsassociated TRAs, as well as complexes of TRA and HLA, may be used toproduce antibodies, using standard techniques well known to thoseskilled in the art.

When "disorder" is used herein, it refers to any pathological conditionwhere the tumor rejection antigen precursor is expressed. An example ofsuch a disorder is cancer melanoma in particular.

Therapeutic approaches based upon the disclosure are premised on aresponse by a subject's immune system, leading to lysis of TRApresenting cells, such as HLA-Cw*1601. One such approach is theadministration of CTLs specific to the complex to a subject withabnormal cells of the phenotype at issue. It is within the skill of theartisan to develop such CTLs in vitro. Specifically, a sample of cells,such as blood cells, are contacted to a cell presenting the complex andcapable of provoking a specific CTL to proliferate. The target cell canbe a transfectant, such as a COS cell of the type described supra. Thesetransfectants present the desired complex on their surface and, whencombined with a CTL of interest, stimulate its proliferation. COS cells,such as those used herein, are widely available, as are other suitablehost cells.

To detail the therapeutic methodology, referred to as adoptive transfer(Greenberg, J. Immunol., 136(5): 1917 (1986); Reddel et al., Science,257: 238 (Jul. 10, 1992); Lynch et al., Eur. J. Immunol., 21: 1403-1410(1991); Kast et al., Cell, 59: 603-614 (Nov. 17, 1989)), cellspresenting the desired complex are combined with CTLs leading toproliferation of the CTLs specific thereto. The proliferated CTLs arethen administered to a subject with a cellular abnormality which ischaracterized by certain of the abnormal cells presenting the particularcomplex. The CTLs then lyse the abnormal cells, thereby achieving thedesired therapeutic goal.

The foregoing therapy assumes that at least some of the subject'sabnormal cells present the relevant HLA/TRA complex. This can bedetermined very easily, as the art is very familiar with methods foridentifying cells which present a particular HLA molecule, as well ashow to identify cells expressing DNA of the pertinent sequences, in thiscase a BAGE sequence. Once cells presenting the relevant complex areidentified via the foregoing screening methodology, they can be combinedwith a sample from a patient, where the sample contains CTLs. If thecomplex presenting cells is lysed by the mixed CTL sample, then it canbe assumed that a BAGE derived, tumor rejection antigen is beingpresented, and the subject is an appropriate candidate for thetherapeutic approaches set forth supra.

Adoptive transfer is not the only form of therapy that is available inaccordance with the invention. CTLs can also be provoked in vivo, usinga number of approaches. One approach, i.e., the use of non-proliferativecells expressing the complex, has been elaborated upon supra. The cellsused in this approach may be those that normally express the complex,such as irradiated melanoma cells or cells transfected with one or bothof the genes necessary for presentation of the complex. Chen et al.,Proc. Natl. Acad. Sci. USA, 88: 110-114 (1991) exemplifies thisapproach, showing the use of transfected cells expressing HPVE7 peptidesin a therapeutic regime. Various cell types may be used. Similarly,vectors carrying one or both of the genes of interest may be used. Viralor bacterial vectors are especially preferred. In these systems, thegene of interest is carried by, for example, a Vaccinia virus or thebacteria BCG, and the materials de facto "infect" host cells. The cellswhich result present the complex of interest, and are recognized byautologous CTLs, which then proliferate. A similar effect can beachieved by combining the tumor rejection antigen or the precursoritself with an adjuvant to facilitate incorporation into HLA-Cw*1601presenting cells which present the HLA molecule of interest. The TRAP isprocessed to yield the peptide partner of the HLA molecule while the TRAis presented without the need for further processing.

Although the invention herein has been described with reference toparticular embodiments, it is to be understood that these embodimentsare merely illustrative of various aspects of the invention. Thus, it isto be understood that numerous modifications may be made in theillustrative embodiments and other arrangements may be devised withoutdeparting from the spirit and scope of the invention.

    __________________________________________________________________________    SEQUENCE LISTING    (1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 9    (2) INFORMATION FOR SEQ ID NO: 1:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 1004    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:    CGCCAATTTAGGGTCTCCGGTATCTCCCGCTGAGCTGCTCTGTTCCCGGCTTAGAGGACC60    AGGAGAAGGGGGAGCTGGAGGCTGGAGCCTGTAACACCGTGGCTCGTCTCACTCTGGATG120    GTGGTGGCAACAGAGATGGCAGCGCAGCTGGAGTGTTAGGAGGGCGGCCTGAGCGGTAGG180    AGTGGGGCTGGAGCAGTAAGATGGCGGCCAGAGCGGTTTTTCTGGCATTGTCT233    MetAlaAlaArgAlaValPheLeuAlaLeuSer    510    GCCCAGCTGCTCCAAGCCAGGCTGATGAAGGAGGAGTCCCCTGTGGTG281    AlaGlnLeuLeuGlnAlaArgLeuMetLysGluGluSerProValVal    152025    AGCTGGAGGTTGGAGCCTGAAGACGGCACAGCTCTGTGCTTCATCTTC329    SerTrpArgLeuGluProGluAspGlyThrAlaLeuCysPheIlePhe    303540    TGAGGTTGTGGCAGCCACGGTGATGGAGACGGCAGCTCAACAGGAGCAATAGGAGGAGAT389    GGAGTTTCACTGTGTCAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCGCCCGCCT449    TGGCCTTCCAAAGTGCCGAGATTACAGCGATGTGCATTTTGTAAGCACTTTGGAGCCACT509    ATCAAATGCTGTGAAGAGAAATGTACCCAGATGTATCATTATCCTTGTGCTGCAGGAGCC569    GGCTCCTTTCAGGATTTCAGTCACATCTTCCTGCTTTGTCCAGAACACATTGACCAAGCT629    CCTGAAAGATGTAAGTTTACTACGCATAGACTTTTAAACTTCAACCAATGTATTTACTGA689    AAATAACAAATGTTGTAAATTCCCTGAGTGTTATTCTACTTGTATTAAAAGGTAATAATA749    CATAATCATTAAAATCTGAGGGATCATTGCCAGAGATTGTTGGGGAGGGAAATGTTATCA809    ACGGTTTCATTGAAATTAAATCCAAAAAGTTATTTCCTCAGAAAAATCAAATAAAGTTTG869    CATGTTTTTTATTCTTAAAACATTTTAAAAACCACTGTAGAATGATGTAAATAGGGACTG929    TGCAGTATTTCTGACATATACTATAAAATTATTAAAAAGTCAATCAGTATTCAACATCTT989    TTACACTAAAAAGCC1004    (2) INFORMATION FOR SEQ ID NO: 2:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 43    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:    MetAlaAlaArgAlaValPheLeuAlaLeuSerAlaGlnLeuLeuGln    51015    AlaArgLeuMetLysGluGluSerProValValSerTrpArgLeuGlu    202530    ProGluAspGlyThrAlaLeuCysPheIlePhe    3540    (2) INFORMATION FOR SEQ ID NO: 3:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:    AlaAlaArgAlaValPheLeuAlaLeu    (2) INFORMATION FOR SEQ ID NO: 4:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:    AlaArgAlaValPheLeuAlaLeuPhe    5    (2) INFORMATION FOR SEQ ID NO:5:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 9    (B) TYPE: amino acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:    MetAlaAlaArgAlaValPheLeuAla    5    (2) INFORMATION FOR SEQ ID NO: 6:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 18    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:    TGGCTCGTCTCACTCTGG18    (2) INFORMATION FOR SEQ ID NO: 7:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:    CCTCCTATTGCTCCTGTTG19    (2) INFORMATION FOR SEQ ID NO: 8:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:    GGCATCGTGATGGACTCCG19    (2) INFORMATION FOR SEQ ID NO: 9:    (i) SEQUENCE CHARACTERISTICS:    (A) LENGTH: 19    (B) TYPE: nucleic acid    (C) STRANDEDNESS: single    (D) TOPOLOGY: linear    (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:    GCTGGAAGGTGGACAGCGA19    __________________________________________________________________________

We claim:
 1. An isolated nucleic acid molecule which hybridizes, understringent conditions, to the nucleotide sequence set forth in SEQ IDNO:1, wherein said isolated nucleic acid molecule consists of anucleotide sequence which codes for a tumor rejection antigen precursor,wherein said isolated nucleic acid molecule does not code for a MAGEtumor rejection antigen precursor.
 2. An isolated molecule which isfully to the nucleic acid molecule of claim 1, wherein said molecule ismRNA or DNA.
 3. A host cell transfected or transformed with the nucleicacid molecule of claim
 1. 4. An expression vector comprising theisolated nucleic acid molecule of claim 1 operably linked to a promoter.5. The host cell of claim 3, wherein said host cell is a mammalian cellwhich expresses HLA-Cw*1601.
 6. The expression vector of claim 4,further comprising a nucleic acid molecule which codes for HLA-Cw*1601.7. An expression kit comprising a separate portion of each of:(i) theisolated nucleic acid molecule of claim 1, and (ii) a nucleic acidmolecule which codes for HLA-Cw*1601.
 8. A host cell transfected ortransformed with the expression vector of claim
 4. 9. A host celltransfected or transformed with the expression vector of claim 6.